TLR9-Independent Activation of B Lymphocytes by Bacterial DNA

Abstract

The intracellular Toll-like receptor 9 (TLR9) is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. Work from this laboratory showed that plasmid DNA is spontaneously internalized in B lymphocytes. This event is followed by the upregulation of costimulatory molecules and the acquisition of antigen presenting function by these cells. However, it is not known whether this phenomenon depends on TLR9. Because of the relevant role played by DNA-based drugs in immunotherapy and vaccination, and the central role of TLR9 signaling by CpG motifs, we decided to investigate whether signaling through TLR9 is a prerequisite for spontaneous transgenesis of lymphocytes. Here we found that transgene expression and upregulation of CD40 and CD86 costimulatory molecules was not inhibited by chloroquine treatment. Spontaneous transgenesis also occurred in B lymphocytes from TLR9-/- mice, and the injection of TLR9-/- transgenic B lymphocytes in C57Bl/6 mice induced both CD4 and CD8 T cell responses comparable to those induced by wild-type B lymphocytes. Collectively, these results suggest that plasmid DNA activates mammalian B lymphocytes through a TLR9 independent pathway.

KDEL-Retained Antigen in B Lymphocytes Induces a Proinflammatory Response: A Possible Role for Endoplasmic Reticulum Stress in Adaptive T Cell Immunity

“…We found that when compared with LPS or CpG ODN, pDNA induced a similar magnitude of proliferation as control ODN (Fig. 3), confirming that pDNA is not mitogenic for B lymphocytes (31,32). In contrast, B lymphocytes incubated with ␥1E␣ 3 KDEL or ␥1E␣ 3 pDNA did up-regulate the costimulatory molecule CD86 (data not shown), another marker of B cell activation.…”

Surface Expression of Toll-Like Receptor 9 Is Upregulated on Intestinal Epithelial Cells in Response to Pathogenic Bacterial DNA

“…However, other workers have described surface expression of TLR9 in various types of cells (6,7,9,18,27). Furthermore, studies using chloroquine to inhibit endosomal acidification have produced different results; some investigators have observed inhibition of TLR9 signaling (1,10,30), and other workers have observed no effect, indicating that there may be an alternate pathway (8,18). In our study, TLR9 was shown by flow cytometry, immunofluorescence, and biotinylation of surface proteins to be expressed on the surface of intestinal epithelial cells.…”

Innate immune recognition of, and regulation by, DNA

“…They further demonstrated that dsDNA-transfected MEFs were protected against HSV infection in vitro. Cortez-Gonzalez et al observed that B cells transfected with ds plasmid DNA upregulated co-stimulatory molecules in a TLR9-independent manner, enabling these B cells to be potent antigen-presenting cells [15]. The physiological role of TLR9-independent recognition of dsDNA has been suggested to be as an endogenous T helper 1 (Th1)-type adjuvant [7,38]; however, roles in infection, autoimmune diseases, allergy and cancer, as well as in DNA-based immunotherapy, should be clarified by future studies.…”

“…Moreover, double-stranded (ds)DNA derived not only from microbes but also from host cells activates the innate immune system in a sequence-independent manner when either it is introduced into the cytosol [6,7] or its homeostatic clearance is hampered [8]. This was not mediated by TLR (9), but instead by an as-yet-undefined DNA recognition machinery that activates both immune and non-immune cells to produce type I interferons (IFNs), cytokines and chemokines, as well as by the upregulation of co-stimulatory molecules [9][10][11][12][13][14][15] (Figure 1). Therefore, during infection and/or tissue damage, DNA derived from pathogens and damaged host cells might regulate both innate and adaptive immune responses.…”